Sample-to-answer microfluidic device towards the point-of-need detection of <i>Staphylococcus aureus</i> enterotoxin genes in ruminant milk

Milk is commonly screened both for indicators of animal disease and health, but also for foodborne hazards. Included in these analyses is the detection of <i>Staphylococcus aureus</i>, that can produce an enterotoxin, causing staphylococcal food poisoning (SFP), which often leads to sudden onset of significant gastrointestinal symptoms in humans. Epidemiological data on SFP are limited, particularly in low- and middle-income countries. Many conventional assays for the detection of staphylococcal enterotoxins rely on the detection of the genes coding for them, either directly in food samples or after bacterial culture. Currently, many of the nucleic acid-based methods used require specific expertise and equipment, whilst bacterial culture takes 24-48 hours; both are contributory factors that limit efforts either during food safety emergencies or routine screening. Here we present the development of a "sample-to-answer" isothermal nucleic acid loop-mediated amplification (LAMP) assay in a microfluidic device for the detection of <i>Staphylococcus aureus</i> enterotoxin genes in ruminant milk. A multiplex LAMP assay targeting two of the most prevalent <i>S. aureus</i> enterotoxin-encoding genes (A and B) was integrated into a microfluidic device combining simple 1 : 10 dilution for sample preparation and a lateral flow assay for easy readout. We achieved a limit of detection of 10⁴ colony forming units per ml in spiked cow and goat milk samples, an order of magnitude more sensitive than the European recommendation for the maximum allowable presence of coagulase-positive staphylococci in raw milk. The assay showed no cross-reactivity in detecting other tested non-enterotoxigenic <i>S. aureus</i> strains or associated foodborne pathogens. The test integrated the simplicity of use of microfluidic devices with the sensitivity, specificity and rapidity of a nucleic acid-based assay, and a simple lateral flow readout to provide an appropriate device to ensure the safety of milk for human consumption. To illustrate its potential for point-of-need practical applications, the test was performed in agricultural settings in rural Turkey in a limited feasibility exercise.